Nuclear Lamina Interactions and Their Role in Gene Repression at the Nuclear Periphery
DOI:
https://doi.org/10.5281/zenodo.22760173Keywords:
lamina-associated domain, LAD, nuclear periphery, DamID, Lamin B1, heterochromatin, gene repressionAbstract
Large chromosomal domains contact the nuclear lamina at the nuclear periphery, forming lamina-associated
domains (LADs) that are broadly associated with transcriptional repression, yet whether peripheral positioning is
itself a repressive cause or a largely correlative consequence of an already-repressed chromatin state has
remained contested. Here we combined DamID-sequencing mapping of Lamin B1 contacts, CRISPR-based forced
tethering of a defined euchromatic locus to the nuclear lamina, and released re-positioning of an endogenous LAD
away from the periphery, in human fibroblasts, to test the causal contribution of peripheral positioning to gene
repression directly. Genome-wide DamID mapping across a proliferation time course identified 1,344 LADs
covering approximately 38% of the genome, with LAD boundaries showing strong, reproducible overlap with
domains of low gene density, late replication timing and H3K9me2 enrichment. Forced tethering of a normally
lamina-distal, actively transcribed 180 kb locus to the nuclear envelope using a doxycycline-inducible
LaminB1-dCas9 tethering system reduced expression of resident genes by 45-72% within 48 hours, without a
detectable change in local DNA methylation, and this repression was substantially, though not completely, reversed
within 72 hours of doxycycline withdrawal and release from the periphery. Conversely, CRISPR-mediated disruption
of a lamina-interaction region within an endogenous LAD reduced Lamin B1 DamID signal at the targeted domain
and produced partial de-repression of resident genes, proportional to the magnitude of lamina-contact loss
achieved at each of three independently targeted interaction regions. These results demonstrate that peripheral
nuclear positioning makes a direct, reversible and quantitatively graded causal contribution to gene repression at
the nuclear periphery, beyond simply marking loci that are repressed for independent chromatin-intrinsic reasons.
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